Oligoribonucleotide assays for novel antibiotics

Abstract

The oligoribonucleotide analogs of the invention are relatively small, three-dimensional structures derived from larger parental RNA molecules. The analogs include a first nucleic acid structure including one or more nucleotide sequences that are derived from a region of parental RNA, wherein in its native state, the region binds to a ligand, e.g., an aminoglycoside, with a parental RNA ligand binding pattern, and a second nucleic acid structure including one or more nucleotide sequences combined with the first nucleic acid structure to form the analog and provide the analog with a conformation that binds the ligand with a ligand binding pattern that is substantially identical to the parental RNA ligand binding pattern. These analogs can be used to identify novel therapeutic compounds.

What is claimed is: 
     
       1. An artificial oligoribonucleotide analog having both natural and heterologous sequences contained therein, wherein said artificial oligoribonucleotide analog has a three dimensional structure that mimics a ligand binding region of a larger parental RNA molecule, said artificial oligoribonucleotide analog comprising; a first oligoribonucleic acid structure whose sequence is identical to the sequence of said ligand binding region; and   a second nucleic acid structure consisting of a heterologous sequence that does not exist adjacent to the sequence of said ligand binding region in said parental RNA molecule; wherein i) the conformation of said second nucleic acid structure is not naturally present adjacent to said ligand binding region;   ii) said second nucleic acid structure stabilizes the conformation of said first oligoribonucleic acid structure so as to mimic the conformation of said parental RNA molecule ligand binding region so that said first oligoribonucleic acid structure binds said ligand with a binding pattern substantially identical to said parental RNA molecule binding pattern; and   iii) said first and second structures are linked by one or more covalent or non-covalent bonds.     
     
     
       2. An artificial oligoribonucleotide analog of claim 1, wherein the sequence of said first nucleic acid structure is identical to a ligand binding region of a 16S ribosomal RNA (rRNA). 
     
     
       3. An artificial oligoribonucleotide analog of claim 2, wherein said ligand binding region comprises the decoding region of 16S rRNA. 
     
     
       4. An artificial oligoribonucleotide analog of claim 3, wherein said ligand binding region comprises nucleotides 1398-1410 and 1490-1505 of 16S rRNA. 
     
     
       5. An artificial oligoribonucleotide analog of claim 3, wherein said ligand binding region comprises the A site subdomain of the decoding region of 16S rRNA. 
     
     
       6. An artificial oligoribonucleotide analog of claim 5, wherein said ligand binding region comprises nucleotides 1404-1410 and 1490-1497 of 16S rRNA. 
     
     
       7. An artificial oligoribonucleotide analog of claim 1, wherein said second nucleic acid structure comprises a tetraloop. 
     
     
       8. An artificial oligoribonucleotide analog of claim 7, wherein said tetraloop comprises the nucleotide sequence:   5'-CCUUCGGG-3'.     
     
     
       9. An artificial oligoribonucleotide analog of claim 1, wherein said second nucleic acid structure comprises two nucleotide sequences forming a based-paired nucleotide clamp. 
     
     
       10. An artificial oligoribonucleotide analog of claim 9, wherein said base-paired nucleotide clamp comprises the nucleotide sequence:   3'-CGUGUC-5'       5'-GCACAG-3'.     
     
     
       11. An artificial oligoribonucleotide analog of claim 9, wherein said base-paired nucleotide clamp comprises the nucleotide sequence:   3'-CC-5'       5'-GG-3'.     
     
     
       12. An artificial oligoribonucleotide analog of claim 1, wherein the sequence of said first oligoribonucleic acid structure is identical to a decoding region of 16S rRNA, and said second nucleic acid structure comprises a tetraloop and a base-paired nucleotide clamp. 
     
     
       13. An artificial oligoribonucleotide analog of claim 12, wherein said decoding region comprises nucleotides 1398-1410 and 1490-1505 of 16S rRNA, said tetraloop comprises the nucleotide sequence 5'-CCUUCGGG-3', said base-paired nucleotide clamp comprises the nucleotide sequence:   3'-CGUGUC-5'       5'-GCACAG-3',     and the complete linear nucleotide sequence of said combined first and second nucleotide structures of said analog is 5'-GCACAGACCGCCCGUCACACCUUCGGGUGAAGUCGUAACAAGGCUGUGC-3' (SEQ ID NO:1).   
     
     
       14. An artificial oligoribonucleotide analog of claim 12, wherein said decoding region comprises nucleotides 1404-1410 and 1490-1497 of 16S rRNA, said tetraloop comprises the nucleotide sequence 5'-CCUUCGGG-3', said base-paired nucleotide clamp comprises the nucleotide sequence:   3'-CC-5'       5'-GG-3',     and the complete linear nucleotide sequence of said combined first and second nucleotide structures of said analog is 5'-GGCGUCACACCUUCGGGUGAAGUCGCC-3' (SEQ ID NO:11).   
     
     
       15. A binding assay for determining the potential therapeutic activity of a test compound, said assay comprising the steps of mixing a test compound with an artificial oligoribonucleotide analog of claim 1 under conditions that allow formation of a binding complex between said analog and said test compound, and   detecting the formation of a binding complex, wherein the presence of a binding complex indicates that said test compound has potential therapeutic activity.   
     
     
       16. A method of claim 15, wherein said artificial oligoribonucleotide analog is labelled and immobilized on a surface, and said binding complex is detected by monitoring changes in the signal of said label when a test compound is bound to said analog. 
     
     
       17. A method of claim 15, wherein said artificial oligoribonucleotide analog is immobilized on a surface, said test compound is labelled, and said binding complex is detected by detecting said label bound to said immobilized analog. 
     
     
       18. A method of claim 16, wherein said artificial oligoribonucleotide analog is fluorescently or radioactively labelled. 
     
     
       19. A competitive binding assay for determining the potential therapeutic activity of a test compound, said assay comprising the steps of mixing an artificial oligoribonucleotide analog of claim 1 with an analog-binding ligand under conditions that allow formation of a first binding complex between said analog and said ligand,   mixing a test compound with the first binding complex under conditions that allow said test compound to disrupt said first binding complex to form a second binding complex between said analog and said test compound, and   detecting the disruption of the first binding complex, wherein the disruption of the first binding complex indicates that said test compound has potential therapeutic activity.   
     
     
       20. A method of claim 19, wherein said ligand is labelled, said artificial oligoribonucleotide analog is immobilized on a surface, and the disruption of the first binding complex is detected by monitoring any decrease in the signal of said label when a test compound displaces said ligand from said first binding complex. 
     
     
       21. A method of claim 19, wherein said artificial oligoribonucleotide analog is labelled, said ligand is immobilized on a surface, and the disruption of the first binding complex is detected by monitoring any decrease in the signal of said label when a test compound displaces said analog from said first binding complex. 
     
     
       22. A method of claim 21, wherein said artificial oligoribonucleotide analog is fluorescently or radioactively labelled. 
     
     
       23. A method of claim 21, wherein said ligand is fluorescently or radioactively labelled. 
     
     
       24. An in situ footprinting assay for determining the potential therapeutic activity of a test compound, said assay comprising the steps of mixing an artificial oligoribonucleotide analog of claim 1 with a test compound under conditions that allow formation of a binding complex between said analog and said test compound,   incubating said binding complex with a chemical probing reagent and monitoring for an effect of said reagent on said analog in said complex,   in a separate control reaction, incubating said analog unbound to any test compound with said chemical probing reagent and monitoring for an effect of said reagent on the unbound analog, and   comparing any effects of said probing reagent on said analog in said binding complex and on said unbound analog, wherein prevention of an effect of said reagent on said analog in said binding complex caused by said reagent on said unbound analog indicates that said test compound has potential therapeutic activity.   
     
     
       25. An in situ footprinting assay of claim 24, wherein said chemical probing reagent is dimethyl sulfate, kethoxal, or carbodiimmide. 
     
     
       26. An in situ footprinting assay of claim 24, wherein said probing reagent covalently modifies a nucleotide in said artificial oligoribonucleotide analog when unbound to any test compound. 
     
     
       27. An in situ footprinting assay of claim 26, wherein the effect of said probing reagent is monitored by use of a labelled oligonucleotide that hybridizes to said artificial oligoribonucleotide analog when protected by said test compound from methylation, and does not hybridize to said artificial oligoribonucleotide analog when methylated by said reagent, the presence of said label after completion of said assay indicating that said test compound has potential therapeutic activity. 
     
     
       28. An in situ footprinting assay of claim 27, wherein said labelled oligonucleotide has the sequence CAGUGU. 
     
     
       29. An in situ footprinting assay of claim 26, wherein the effect of said probing reagent is monitored by use of an oligonucleotide primer that is complementary to a portion of said artificial oligoribonucleotide analog, a labelled nucleotide, and reverse transcriptase, wherein extension of said primer on said analog with said labelled nucleotide does not occur when said artificial oligoribonucleotide analog is methylated by said reagent, the presence of said label after completion of said assay indicating that said test compound has potential therapeutic activity. 
     
     
       30. An in situ footprinting assay of claim 29, wherein said oligonucleotide primer has the sequence TTCACCCGGAAGGTG (SEQ ID NO:12). 
     
     
       31. An analog of claim 1, wherein said ligand is an aminoglycoside.